Methods and compositions for the prevention and treatment neuropathy

ABSTRACT

The disclosure relates to methods for treating a subject suffering from hyperalgesia caused by drug-induced neuropathy by administering to the subject an effective amount of an aromatic-cationic peptide. The disclosure also relates to methods for protecting a subject from hyperalgesia caused by drug-induced neuropathy by administering an effective amount of an aromatic-cationic peptide to a subject at risk for developing the condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/488,697, filed on Apr. 17, 2017, now U.S. Pat. No. 10,279,008, whichis a Continuation of U.S. patent application Ser. No. 14/388,705, filedon Sep. 26, 2014, now U.S. Pat. No. 9,636,378, which is the U.S.National Stage of International Application No. PCT/US2013/034647, filedon Mar. 29, 2013, which claims priority to U.S. Provisional ApplicationNo. 61/618,428, filed Mar. 30, 2012, the contents of which are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

The present technology relates generally to compositions for preventingor treating neuropathy comprising an aromatic-cationic peptide andmethods for using the same.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or the references cited areadmitted to be prior art to the present technology.

Chemotherapy-induced peripheral neuropathy results in patient sufferingand limits the scope treatment with potentially useful anti-cancerdrugs. Vinca alkaloids, such as vincristine and vinblastine, havesignificant efficacy in the treatment of malignant tumors. However,these agents are highly neurotoxic, frequently causing painfulperipheral neuropathies that limit the dose and duration of use incancer treatment. These side effects are dose and time dependent, oftenwith latencies of days to weeks, and may be cumulative with toxicitiesassociated with other drugs used in combination with chemotherapeuticagents.

SUMMARY

The present disclosure provides compositions and methods for treating orpreventing neuropathy or hyperalgesia comprising administering anaromatic-cationic peptide to a subject in need thereof.

In one aspect, the present disclosure provides a method for treatingperipheral neuropathy or hyperalgesia in a subject in need thereof,comprising administering to the subject an effective amount of a peptidehaving the formula D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂. In someembodiments, the peripheral neuropathy or hyperalgesia is drug-induced.

In some embodiments, the drug is a chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is procarbazine, nitrofurazone,podophyllum, mustine, ethoglucid, cisplatin, suramin, paclitaxel,chlorambucil, altretamine, carboplatin, cytarabine, docetaxel,dacarbazine, etoposide, ifosfamide with mesna, fludarabine, tamoxifen,teniposide, thioguanine, or vincristine. In some embodiments, thechemotherapeutic agent is vincristine.

In some embodiments, the peptide is administered simultaneous with thedrug. In some embodiments, the peptide is administered subsequent to thedrug. In some embodiments, the peripheral neuropathy causeshyperalgesia. In some embodiments, the subject is a human. In someembodiments, the peptide is administered intravenously, orally,subcutaneously, transdermally, intraperitoneally, or topically.

In one aspect, the present disclosure provides a method for preventinghyperalgesia in a subject in need thereof, comprising administering tothe subject an effective amount of a peptide having the formulaD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂. In some embodiments, thehyperalgesia is drug-induced. In some embodiments, the drug is achemotherapeutic agent.

In some embodiments, the chemotherapeutic agent is procarbazine,nitrofurazone, podophyllum, mustine, ethoglucid, cisplatin, suramin,paclitaxel, chlorambucil, altretamine, carboplatin, cytarabine,docetaxel, dacarbazine, etoposide, ifosfamide with mesna, fludarabine,tamoxifen, teniposide, thioguanine, or vincristine. In some embodiments,the chemotherapeutic agent is vincristine.

In some embodiments, the peptide is administered simultaneous with thedrug. In some embodiments, the peptide is administered subsequent to thedrug. In some embodiments, the peptide is administered prior the onsetof hyperalgesia. In some embodiments, the subject is a human. In someembodiments, the peptide is administered intravenously, orally,subcutaneously, transdermally, intraperitoneally, intrathecallyintramuscularly, intranasally, bucally, sublingually, translingually, ortopically.

In one aspect, the present disclosure provides a composition fortreating or preventing hyperalgesia in a subject in need thereof,comprising an effective amount of a peptide having the formulaD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂. In some embodiments, thehyperalgesia is drug-induced.

In some embodiments, the drug is a chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is procarbazine, nitrofurazone,podophyllum, mustine, ethoglucid, cisplatin, suramin, paclitaxel,chlorambucil, altretamine, carboplatin, cytarabine, docetaxel,dacarbazine, etoposide, ifosfamide with mesna, fludarabine, tamoxifen,teniposide, thioguanine, or vincristine. In some embodiments, thechemotherapeutic agent is vincristine.

In some embodiments, the peptide is administered simultaneous with thedrug. In some embodiments, the peptide is administered subsequent to thedrug. In some embodiments, the peptide is administered prior the onsetof hyperalgesia. In some embodiments, the subject is a human. In someembodiments, the peptide is administered intravenously, orally,subcutaneously, transdermally, intraperitoneally, intrathecallyintramuscularly, intranasally, bucally, sublingually, translingually, ortopically.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

In one embodiment, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R⁸, and R⁹ are all hydrogen; and n is 4.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart showing that D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂is effective in the prevention of vincristine-induced hyperalgesia inrats. # p>0.05 for the comparison of Groups 1 and 3; **p<0.01 for thecomparison of Groups 1 and A.

FIG. 2 is a chart showing that D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂is effective in the treatment of vincristine-induced hyperalgesia inrats.

DETAILED DESCRIPTION

The present disclosure is based on the surprising discovery that certainaromatic-cationic peptides can treat or ameliorate neuropathy andhyperalgesia, including drug-induced neuropathy and hyperalgesia. It isto be appreciated that certain aspects, modes, embodiments, variations,and features of the present technology are described below in variouslevels of detail in order to provide a substantial understanding of thepresent technology.

In practicing the present technology, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which the present technologybelongs.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the enumerated value.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),rectally, or topically. Administration includes self-administration andthe administration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in, aneuropathy or one or more conditions associated with a neuropathy,including, but not limited to, hyperalgesia. In the context oftherapeutic or prophylactic applications, the amount of a compositionadministered to the subject will depend on the type and severity of theneuropathy or hyperalgesia and on the characteristics of the individual,such as general health, age, sex, body weight and tolerance to drugs. Itwill also depend on the degree, severity, and type of the neuropathy orhyperalgesia. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. The compositions can alsobe administered in combination with one or more additional therapeuticcompounds.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with therapeutic uses of the agent. Suchinterfering materials may include enzymes, hormones and otherproteinaceous and nonproteinaceous solutes.

As used herein, the term “medical condition” includes, but is notlimited to, any condition or disease manifested as one or more physicaland/or psychological symptoms for which treatment and/or prevention isdesirable, and includes previously and newly identified diseases andother disorders. For example, a medical condition may be a neuropathy,or any associated conditions or complications, including, but notlimited to, hyperalgesia.

As used herein, “neuropathy” or “peripheral neuropathy” refers generallyto damage to nerves of the peripheral nervous system. The termencompasses neuropathy of various etiologies, including, but notlimited, to neuropathy caused by, resulting from, or associated withgenetic disorders, metabolic/endocrine complications, diabetes,inflammatory diseases, vitamin deficiencies, malignant diseases, andtoxicity, such as alcohol, organic metal, heavy metal, radiation, anddrug toxicity. As used herein, the term encompasses motor, sensory,mixed sensorimotor, chronic, and acute neuropathy. As used herein theterm encompasses mononeuropathy, multiple mononeuropathy, andpolyneuropathy.

Illustrative causes of neuropathy include, but are not limited to,neuropathy caused by, resulting from, or associated with diabetes,chemotherapy, trauma, malnutrition, alcoholism, autoimmune diseases,cancer, infectious diseases, kidney disease, liver disease, HIV, AIDS,hypothyroidism, hereditary disorders, and exposure to toxins.

In some embodiments, the present disclosure provides compositions forthe treatment or prevention of peripheral neuropathy or the symptoms ofperipheral neuropathy. In some embodiments, the peripheral neuropathy isdrug-induced peripheral neuropathy. In some embodiments, the peripheralneuropathy is induced by a chemotherapeutic agent. In some embodiments,the chemotherapeutic agent is a vinca alkaloid. In some embodiments, thevinca alkaloid is vincristine. In some embodiments, the symptoms ofperipheral neuropathy include hyperalgesia.

As used herein, “hyperalgesia” refers to an increased sensitivity topain, which may be caused by damage to nociceptors or peripheral nerves(i.e. neuropathy). The term refers to temporary and permanenthyperalgesia, and encompasses both primary hyperalgesia (i.e. painsensitivity occurring directly in damaged tissues) and secondaryhyperalgesia (i.e. pain sensitivity occurring in undamaged tissuessurrounding damaged tissues). The term encompasses hyperalgesia causedby, but not limited to, neuropathy caused by, resulting from, orotherwise associated with genetic disorders, metabolic/endocrinecomplications, inflammatory diseases, vitamin deficiencies, malignantdiseases, and toxicity, such as alcohol, organic metal, heavy metal,radiation, and drug toxicity. In some embodiments hyperalgesia is causedby drug-induced peripheral neuropathy.

In some embodiments, the present disclosure provides compositions forthe treatment or prevention of hyperalgesia. In some embodiments, thehyperalgesia is drug-induced. In some embodiments, the hyperalgesia isinduced by a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is a vinca alkaloid. In some embodiments, thevinca alkaloid is vincristine.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts, as well as in avoluminous research literature.

As used herein, “prevention” or “preventing” of a disorder or conditionwith reference to a treatment method (e.g., administration of acompound, such as an aromatic-cationic peptide of the presentdisclosure), means that the method reduces the occurrence of thedisorder or condition in treated subjects relative to an untreatedcontrol subjects.

As used herein, the words “protect” or “protecting” refer to decreasingthe likelihood and/or risk that the subject treated with a peptide ofthe present technology will develop a given disease or disorder, ordelaying the onset or reducing the severity of one or more symptoms ofthe disease, disorder or condition, e.g., a neuropathy or associatedconditions or complications such as hyperalgesia. Typically, thelikelihood of developing the disease or disorder is considered to bereduced if the likelihood is decreased by at least about 10%, at leastabout 25%, at least about 50%, at least about 75%, at least about 90%,in comparison to the likelihood and/or risk that the same subjectuntreated with a peptide of the present technology will develop aneuropathy or a hyperalgesia. In some embodiments, the peptides protecta subject against the development of a neuropathy or a hyperalgesia whenthe peptides are administered after a subject receives aneuropathy-inducing drug, but before the onset of neuropathy orhyperalgesia.

The term “subject” as used herein refers to a member of any vertebratespecies. The methods of the presently disclosed subject matter areparticularly useful for warm-blooded vertebrates. Provided herein is thetreatment of mammals such as humans, as well as those mammals ofimportance due to being endangered, of economic importance (animalsraised on farms for consumption by humans) and/or social importance(animals kept as pets or in zoos) to humans. In some embodiments, thesubject is a human.

As used herein, the terms “treating,” “treatment,” or “alleviation”refer to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for a disease or condition if, after receiving a therapeuticamount of the aromatic-cationic peptides according to the methodsdescribed herein, the subject shows observable and/or measurablereduction in or absence of one or more signs and symptoms of aparticular disease or condition. For example, for a neuropathy or ahyperalgesia, treatment or prevention may include a reduction frequency,severity, or duration of symptoms, such as pain in the extremities. Itis also to be appreciated that the various modes of treatment orprevention of medical conditions as described are intended to mean“substantial”, which includes total but also less than total treatmentor prevention, and wherein some biologically or medically relevantresult is achieved.

Peptides

The aromatic-cationic peptides useful in the present methods arewater-soluble and highly polar. Despite these properties, the peptidescan readily penetrate cell membranes. The aromatic-cationic peptidesuseful in the present methods include a minimum of three amino acids,and preferably include a minimum of four amino acids, covalently joinedby peptide bonds. The maximum number of amino acids present in thearomatic-cationic peptides of the present methods is about twenty aminoacids covalently joined by peptide bonds. In some embodiments, themaximum number of amino acids is about twelve, about nine, or about six.In some embodiments, the number of amino acids present in the peptidesis four.

The amino acids of the aromatic-cationic peptides can be any amino acid.The amino acids may be naturally occurring. Naturally occurring aminoacids include, for example, the twenty most common levorotatory (L)amino acids normally found in mammalian proteins, i.e., alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val). Other naturallyoccurring amino acids include, for example, amino acids that aresynthesized in metabolic processes not associated with proteinsynthesis. For example, the amino acids ornithine and citrulline aresynthesized in mammalian metabolism during the production of urea.

The peptides can optionally contain one or more non-naturally occurringamino acids. The non-naturally occurring amino acids may be L-,dextrorotatory (D), or mixtures thereof. The peptide may have no aminoacids that are naturally occurring. Non-naturally occurring amino acidsare those amino acids that typically are not synthesized in normalmetabolic processes in living organisms, and do not naturally occur inproteins. In addition, the non-naturally occurring amino acids are notrecognized by common proteases.

The non-naturally occurring amino acid can be present at any position inthe peptide. For example, the non-naturally occurring amino acid can beat the N-terminus, the C-terminus, or at any position between theN-terminus and the C-terminus. The non-natural amino acids may, forexample, comprise alkyl, aryl, or alkylaryl groups. Some examples ofalkyl amino acids include α-aminobutyric acid, β-aminobutyric acid,γ-aminobutyric acid, δ-aminovaleric acid, and ε-aminocaproic acid. Someexamples of aryl amino acids include ortho-, meta, and para-aminobenzoicacid. Some examples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivatives of naturally occurringamino acids may, for example, include the addition of one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of a modification of an amino acid is the derivatizationof a carboxyl group of an aspartic acid or a glutamic acid residue ofthe peptide. One example of derivatization is amidation with ammonia orwith a primary or secondary amine, e.g., methylamine, ethylamine,dimethylamine or diethylamine. Another example of derivatizationincludes esterification with, for example, methyl or ethyl alcohol.Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group.

The non-naturally occurring amino acids are suitably resistant, and insome embodiments, insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD- non-naturally occurring amino acids. The D-amino acids do notnormally occur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides may have lessthan five, less than four, less than three, less than two contiguousL-amino acids recognized by common proteases, irrespective of whetherthe amino acids are naturally or non-naturally occurring. If the peptidecontains protease sensitive sequences of amino acids, at least one ofthe amino acids may be a non-naturally-occurring D-amino acid, therebyconferring protease resistance. An example of a protease sensitivesequence includes two or more contiguous basic amino acids that arereadily cleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

In suitable embodiments, the aromatic-cationic peptides have a minimumnumber of net positive charges at physiological pH in comparison to thetotal number of amino acid residues in the peptide. The minimum numberof net positive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid. Typically, a peptide has apositively charged N-terminal amino group and a negatively chargedC-terminal carboxyl group. The charges cancel each other out atphysiological pH.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, preferably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges. In suitable embodiments, the aromatic-cationic peptides have aminimum number of aromatic groups in comparison to the total number ofnet positive charges (p_(t)). The minimum number of aromatic groups willbe referred to below as (a).

Naturally occurring amino acids that have an aromatic group include theamino acids histidine, tryptophan, tyrosine, and phenylalanine. Forexample, the hexapeptide Lys-Gln-Tyr-D-Arg-Phe-Trp has a net positivecharge of two (contributed by the lysine and arginine residues) andthree aromatic groups (contributed by tyrosine, phenylalanine andtryptophan residues).

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges at physiological pH (p_(t)) wherein 3a is thelargest number that is less than or equal to p_(t)+1, except that whenp_(t) is 1, a may also be 1. In this embodiment, the relationshipbetween the minimum number of aromatic groups (a) and the total numberof net positive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, may be amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group.

The free carboxylate groups of the asparagine, glutamine, aspartic acid,and glutamic acid residues not occurring at the C-terminus of thearomatic-cationic peptides of the present technology may also beamidated wherever they occur within the peptide. The amidation at theseinternal positions may be with ammonia or any of the primary orsecondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide havingtwo net positive charges and at least one aromatic amino acid. In aparticular embodiment, the aromatic-cationic peptide is a tripeptidehaving two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, thefollowing exemplary peptides:

-   -   Lys-D-Arg-Tyr-NH₂    -   Phe-D-Arg-His    -   D-Tyr-Trp-Lys-NH₂    -   Trp-D-Lys-Tyr-Arg-NH₂    -   Tyr-His-D-Gly-Met    -   Phe-Arg-D-His-Asp    -   Tyr-D-Arg-Phe-Lys-Glu-NH₂    -   Met-Tyr-D-Lys-Phe-Arg    -   D-His-Glu-Lys-Tyr-D-Phe-Arg    -   Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂    -   Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His    -   Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂    -   Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂    -   Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys    -   Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂    -   Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys    -   Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂    -   D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂    -   Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe    -   Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe    -   Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂    -   Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr    -   Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys    -   Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂    -   Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly    -   D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂    -   Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe    -   His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂    -   Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp    -   Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

In some embodiments, peptides are those peptides which have a tyrosineresidue or a tyrosine derivative. Suitable derivatives of tyrosineinclude 2′-methyltyrosine (Mmt); 2′,6′-dimethyltyrosine (2′6′Dmt);3′,5′-dimethyltyrosine (3′5′Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, the peptide has the formula Tyr-D-Arg-Phe-Lys-NH₂.Tyr-D-Arg-Phe-Lys-NH₂ has a net positive charge of three, contributed bythe amino acids tyrosine, arginine, and lysine and has two aromaticgroups contributed by the amino acids phenylalanine and tyrosine. Thetyrosine of Tyr-D-Arg-Phe-Lys-NH₂ can be a modified derivative oftyrosine such as in 2′,6′-dimethyltyrosine to produce the compoundhaving the formula 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂.

In a suitable embodiment, the amino acid residue at the N-terminus isarginine. An example of such a peptide is D-Arg-2′6′Dmt-Lys-Phe-NH₂. Inanother embodiment, the amino acid at the N-terminus is phenylalanine orits derivative. Derivatives of phenylalanine include2′-methylphenylalanine (Mmp), 2′,6′-dimethylphenylalanine (Dmp),N,2′,6′-trimethylphenylalanine (Tmp), and2′-hydroxy-6′-methylphenylalanine (Hmp). An example of such a peptide isPhe-D-Arg-Phe-Lys-NH₂. In one embodiment, the amino acid sequence of2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ is rearranged such that Dmt is not at theN-terminus. An example of such an aromatic-cationic peptide has theformula D-Arg-2′6′Dmt-Lys-Phe-NH₂.

In yet another embodiment, the aromatic-cationic peptide has the formulaPhe-D-Arg-Dmt-Lys-NH₂. Alternatively, the N-terminal phenylalanine canbe a derivative of phenylalanine such as 2′,6′-dimethylphenylalanine(2′6′Dmp). Tyr-D-Arg-Phe-Lys-NH₂ containing 2′,6′-dimethylphenylalanineat amino acid position one has the formula 2′,6′-Dmp-D-Arg-Dmt-Lys-NH₂.

Suitable substitution variants of the peptides include conservativeamino acid substitutions. Amino acids may be grouped according to theirphysicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group are generally more likely to alter thecharacteristics of the original peptide. Examples of peptides include,but are not limited to, the aromatic-cationic peptides shown in Table 5.

TABLE 5 Examples of Aromatic-Cationic Peptides C-Ter- Amino Amino AminoAmino minal Acid Acid Acid Acid Modifi- Position 1 Position 2 Position 3Position 4 cation D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂ D-Arg PheLys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-Arg Lys PheDmt NH₂ D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Lys PheNH₂ D-Arg Dmt Lys Phe NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂Phe D-Arg Phe Lys NH₂ Phe D-Arg Phe Lys NH₂ Phe D-Arg Phe Lys NH₂ PheD-Arg Phe Lys NH₂ Phe D-Arg Phe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-ArgDmt Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg DmtLys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-Arg Lys NH₂ Phe Dmt Lys D-ArgNH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-Arg NH₂ Lys Dmt D-Arg Phe NH₂Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂ Lys D-Arg Dmt Phe NH₂ D-ArgDmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂ D-Arg Dmt D-Arg Tyr NH₂ D-ArgDmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂ Trp D-Arg Tyr Lys NH₂ Trp D-ArgTrp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-Arg Trp Lys Phe NH₂ D-Arg Trp PheLys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg Trp Dmt Lys NH₂ D-Arg Lys Trp PheNH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg Phe Lys NH₂ Ala D-Arg Phe Lys NH₂Cha = cyclohexylalanine

Under certain circumstances, it may be advantageous to use a peptidethat also has opioid receptor agonist activity. Examples of mu-opioidanalogs include, but are not limited to, the aromatic-cationic peptidesshown in Table 6.

TABLE 6 Aromatic-Cationic Peptides with Opioid Receptor Agonist ActivityC-Ter- Amino Amino Amino Amino minal Acid Acid Acid Acid Modifi-Position 1 Position 2 Position 3 Position 4 cation Tyr D-Arg Phe Lys NH₂Tyr D-Arg Phe Orn NH₂ Tyr D-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ TyrD-Arg Phe Lys NH₂ 2′6′Dmt D-Arg Phe Lys NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys- NH₂ NH(CH₂)₂—NH-dns 2′6′Dmt D-Arg Phe Lys- NH₂NH(CH₂)₂—NH-atn 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit Phe Lys NH₂2′6′Dmt D-Cit Phe Lys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg PheOrn NH₂ 2′6′Dmt D-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′DmtD-Arg Phe Ahp(2- NH₂ aminoheptanoic acid) Bio-2′6′Dmt D-Arg Phe Lys NH₂3′5′Dmt D-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg PheDab NH₂ 3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg TyrOrn NH₂ Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg TyrLys NH₂ 2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′DmtD-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′DmtOrn NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′DmtD-Arg 3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ 2′6′Dmt D-Arg2′6′Dmt Lys NH₂ Tyr D-Lys Phe Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′Dmt D-LysPhe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys Phe Dab NH₂3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Lys PheArg NH₂ Tyr D-Lys Tyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr DabNH₂ Tyr D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys TyrOrn NH₂ 2′6′Dmt D-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′DmtD-Lys 2′6′Dmt Lys NH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys2′6′Dmt Dab NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDapNH₂ 2′6′Dmt D-Arg Phe atnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′DmtD-Lys 3′5′Dmt Orn NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys3′5′Dmt Dap NH₂ Tyr D-Lys Phe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-DabPhe Arg NH₂ Tyr D-Dap Phe Arg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′DmtD-Lys Phe Arg NH₂ 2′6′Dmt D-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂3′5′Dmt D-Dap Phe Arg NH₂ 3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys PheArg NH₂ 3′5′Dmt D-Orn Phe Arg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn TyrArg NH₂ Tyr D-Dab Tyr Arg NH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys 2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt ArgNH₂ 2′6′Dmt D-Dab 2′6′Dmt Arg NH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn3′5′Dmt Arg NH₂ Mmt D-Arg Phe Lys NH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-ArgPhe Dab NH₂ Mmt D-Arg Phe Dap NH₂ Tmt D-Arg Phe Lys NH₂ Tmt D-Arg PheOrn NH₂ Tmt D-Arg Phe Dab NH₂ Tmt D-Arg Phe Dap NH₂ Hmt D-Arg Phe LysNH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-Arg Phe Dab NH₂ Hmt D-Arg Phe Dap NH₂Mmt D-Lys Phe Lys NH₂ Mmt D-Lys Phe Orn NH₂ Mmt D-Lys Phe Dab NH₂ MmtD-Lys Phe Dap NH₂ Mmt D-Lys Phe Arg NH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-LysPhe Orn NH₂ Tmt D-Lys Phe Dab NH₂ Tmt D-Lys Phe Dap NH₂ Tmt D-Lys PheArg NH₂ Hmt D-Lys Phe Lys NH₂ Hmt D-Lys Phe Orn NH₂ Hmt D-Lys Phe DabNH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-Lys Phe Arg NH₂ Mmt D-Lys Phe Arg NH₂Mmt D-Orn Phe Arg NH₂ Mmt D-Dab Phe Arg NH₂ Mmt D-Dap Phe Arg NH₂ MmtD-Arg Phe Arg NH₂ Tmt D-Lys Phe Arg NH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-DabPhe Arg NH₂ Tmt D-Dap Phe Arg NH₂ Tmt D-Arg Phe Arg NH₂ Hmt D-Lys PheArg NH₂ Hmt D-Orn Phe Arg NH₂ Hmt D-Dab Phe Arg NH₂ Hmt D-Dap Phe ArgNH₂ Hmt D-Arg Phe Arg NH₂ Dab = diaminobutyric Dap = diaminopropionicacid Dmt = dimethyltyrosine Mmt = 2′-methyltyrosine Tmt =N,2′,6′-trimethyltyrosine Hmt = 2′-hydroxy,6′-methyltyrosine dnsDap =β-dansyl-L-α,β-diaminopropionic acid atnDap =β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

The amino acids of the peptides shown in Tables 5 and 6 may be in eitherthe L- or the D-configuration.

Synthesis of the Peptides

The peptides useful in the methods of the present technology may bechemically synthesized by any of the methods well known in the art.Suitable methods for synthesizing the protein include, for example thosedescribed by Stuart and Young in “Solid Phase Peptide Synthesis,” SecondEdition, Pierce Chemical Company (1984), and in “Solid Phase PeptideSynthesis,” Methods Enzymol., 289, Academic Press, Inc, New York (1997).

Neuropathy and Hyperalgesia

The aromatic-cationic peptides described herein are useful in treatingor preventing neuropathy or hyperalgesia. In some embodiments, thearomatic-cationic peptides may be administered to a subject followingthe onset of neuropathy or hyperalgesia. Thus, the term “treatment” isused herein in its broadest sense and refers to use of anaromatic-cationic peptide for a partial or complete cure of theneuropathy or hyperalgesia, a reduction or amelioration of signs orsymptoms, and/or a reduction of severity of signs or symptoms.

In other embodiments, the aromatic-cationic peptides of the presenttechnology may be administered to a subject before the onset ofneuropathy or hyperalgesia in order to prevent, protect against and/orprovide prophylaxis for neuropathy or hyperalgesia. It is alsocontemplated that the compounds may be administered to a subject at riskof developing neuropathy or hyperalgesia.

The term “peripheral neuropathy” refers generally to damage to nerves ofthe peripheral nervous system. The term encompasses neuropathy ofvarious etiologies, including, but not limited to, acquiredneuropathies, hereditary neuropathies, and idiopathic neuropathies.Illustrative acquired neuropathies include, but are not limited to,e.g., neuropathies caused by, resulting from, or otherwise associatedwith trauma, metabolic/endocrine disorders (e.g., diabetes),inflammatory diseases, infectious diseases, vitamin deficiencies,malignant diseases, and toxicity, such as alcohol, organic metal, heavymetal, radiation, and drug toxicity. As used herein, the “peripheralneuropathy” encompasses motor, sensory, mixed sensorimotor, chronic, andacute neuropathy. As used herein the term encompasses mononeuropathy,multiple mononeuropathy, and polyneuropathy.

Illustrative causes of neuropathy include, but are not limited to,neuropathy caused by, resulting from, or associated with diabetes,chemotherapy, trauma, malnutrition, alcoholism, autoimmune diseases,cancer, infectious diseases, kidney disease, liver disease, HIV, AIDS,hypothyroidism, hereditary disorders, and exposure to toxins.

Drug toxicity causes multiple forms of peripheral neuropathy, with themost common being axonal degeneration. A notable exception is that ofperhexiline, a prophylactic anti-anginal agent that can cause segmentaldemyelination, a localized degeneration of the insulating layer aroundsome nerves.

Peripheral neuropathies usually present sensory symptoms initially, andoften progress to motor disorders. Most drug-induced peripheralneuropathies are purely sensory or mixed sensorimotor defects. Anexception is that of Dapzone, which causes an almost exclusively motorneuropathy.

Drug-induced peripheral neuropathy, including, for example,chemotherapy-induced peripheral neuropathy can cause a variety ofdose-limiting neuropathic conditions, including 1) myalgias, 2) painfulburning paresthesis, 3) glove-and-stocking sensory neuropathy, and 4)hyperalgia and allodynia. Hyperalgia refers to hypersensitivity and paincaused by stimuli that is normally only mildly painful or irritating.Allodynia refers to hypersensitivity and pain caused by stimuli that isnormally not painful or irritating.

The term “hyperalgesia” refers to an increased sensitivity to pain,which may be caused by damage to nociceptors or peripheral nerves (i.e.neuropathy). The term refers to temporary and permanent hyperalgesia,and encompasses both primary hyperalgesia (i.e. pain sensitivityoccurring directly in damaged tissues) and secondary hyperalgesia (i.e.pain sensitivity occurring in undamaged tissues surrounding damagedtissues). The term encompasses hyperalgesia caused by peripheralneuropathy, including, but not limited to, neuropathy caused by,resulting from, or associated with genetic disorders,metabolic/endocrine complications, inflammatory diseases, vitamindeficiencies, malignant diseases, and toxicity, such as alcohol, organicmetal, heavy metal, radiation, and drug toxicity. In some embodimentshyperalgesia is caused by drug-induced peripheral neuropathy.

In some embodiments, the present disclosure provides compositions forthe treatment or prevention of hyperalgesia. In some embodiments, thehyperalgesia is drug-induced. In some embodiments, the hyperalgesia isinduced by a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is a vinca alkaloid. In some embodiments, thevinca alkaloid is vincristine.

A wide variety of pharmaceuticals are known to cause drug-inducedneuropathy, including, but not limited to, e.g., anti-microbials,anti-neoplastic agents, cardiovascular drugs, hypnotics andpsychotropics, anti-rheumatics, and anti-convulsants.

Illustrative anti-microbials known to cause neuropathy include, but arenot limited to, e.g., isoniazid, ethambutol, ethionamide,nitrofurantoin, metronidazole, ciprofloxacin, chloramphenicol,thiamphenicol, diamines, colistin, streptomycin, nalidixic acid,clioquinol, sulphonamides, amphotericin, penicillin.

Illustrative anti-neoplastic agents known to cause neuropathy include,but are not limited to, e.g., procarbazine, nitrofurazone, podophyllum,mustine, ethoglucid, cisplatin, suramin, paclitaxel, chlorambucil,altretamine, carboplatin, cytarabine, docetaxel, dacarbazine, etoposide,ifosfamide with mesna, fludarabine, tamoxifen, teniposide, andthioguanine. Vinca alkaloids, such as vincristine, are known to beparticularly neurotoxic.

Illustrative cardiovascular drugs known to cause neuropathy include, butare not limited to, e.g., propranolol, perhexiline, hydrallazine,amiodarone, disopyramide, and clofibrate.

Illustrative hypnotics and psychotropics known to cause neuropathyinclude, but are not limited to, e.g., phenelzine, thalidomide,methaqualone, glutethimide, amitriptyline, and imipramine.

Illustrative anti-rheumatics known to cause neuropathy include, but arenot limited to, e.g., gold, indomethacin, colchicine, chloroquine, andphenyl butazone.

Illustrative anti-convulsants known to cause neuropathy include, but arenot limited to, e.g., phenytoin.

Other drugs known to cause neuropathy include, but are not limited to,e.g., calcium carbimide, sulfoxone, ergotamine, propylthiouracil,sulthaime, chlorpropamide, methysergide, phenytoin, disulfiram,carbutamide, tolbutamide, methimazole, dapsone, and anti-coagulants.

Methods of Prevention and Treatment

General. The aromatic-cationic peptides described herein are useful toprevent, ameliorate, or treat disease. Specifically, the disclosureprovides for methods of treating or preventing neuropathy orhyperalgesia, comprising administering to a subject in need thereof aneffective amount of an aromatic-cationic peptide. In some embodiments,the peptide is D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂. In someembodiments, the neuropathy or hyperalgesia is drug-induced. In someembodiments, the drug-induced neuropathy or hyperalgesia is caused byadministration of a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is a vinca alkaloid. In some embodiments, thevinca alkaloid is vincristine.

Determination of the Biological Effect of the Aromatic-CationicPeptide-Based Therapeutic.

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific aromatic-cationicpeptide-based therapeutic and whether its administration is indicatedfor treatment of neuropathy or hyperalgesia. In various embodiments, invitro assays can be performed with representative cells of the type(s)involved in the subject's disorder, to determine whether a givenaromatic-cationic peptide-based therapeutic exerts the desired effectupon the cell type(s). Compounds for use in therapy can be tested insuitable animal model systems including, but not limited to, rats, mice,chicken, cows, monkeys, rabbits, and the like, prior to testing in humansubjects. Similarly, for in vivo testing, any of the animal model systemknown in the art can be used prior to administration to human subjects.Conditions associated with neuropathy or hyperalgesia, and the efficacyof aromatic-cationic peptides in preventing or treating such, can bereadily detected, for example, by measuring the sensitivity of animalsubjects to pain stimuli, such as by the methods outlined in Examples 1and 2 below.

Prophylactic Methods.

In one aspect, the present technology provides a method for preventingneuropathy or hyperalgesia in a subject, or symptoms associated withneuropathy or hyperalgesia comprising administering to the subject aneffective amount of an aromatic-cationic peptide such asD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂. In some embodiments, thepeptide is administered simultaneous with one or more therapeutic drugsin order to treat or prevent drug-induced neuropathy or hyperalgesia. Insome embodiments, the peptide is administered prior to one or moretherapeutic drugs in order to treat or prevent drug-induced neuropathyor hyperalgesia. In some embodiments the peptide is administered priorto, and/or simultaneous with, one or more therapeutic drugs in order totreat or prevent drug-induced neuropathy or hyperalgesia. In someembodiments, the peptide is administered simultaneous to and/orsubsequent to the drug in order to treat or prevent neuropathy orhyperalgesia. In some embodiments, the peptide is administeredsubsequent to the administration of one or more therapeutic agents, butprior to the onset of neuropathy or hyperalgesia or symptoms ofneuropathy or hyperalgesia.

Subjects at risk for drug-induced neuropathy or hyperalgesia may beidentified by various diagnostic or prognostic methods known in the art.For example, subjects with a history of drug-induced neuropathy orhyperalgesia, or a family history of neuropathy or hyperalgesia, may beassessed as being at risk for the development of neuropathy orhyperalgesia. Additionally or alternatively, subjects administeredmultiple therapeutic agents, or administered one or more therapeuticagents for an extended period of time, may be assessed as being at riskfor the development of neuropathy or hyperalgesia.

Therapeutic Methods.

Another aspect of the technology includes methods of reducing thesymptoms associated with neuropathy or hyperalgesia in a subject fortherapeutic purposes. In therapeutic applications, compositions ormedicaments are administered to a subject suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease, including itscomplications and intermediate pathological phenotypes in development ofthe disease. As such, the present technology provides methods oftreating an individual afflicted with neuropathy or hyperalgesia orsymptoms associated with neuropathy or hyperalgesia.

In one embodiment, administration of an aromatic-cationic peptide suchas D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ to a subject exhibiting oneor more symptoms of neuropathy or hyperalgesia will cause an improvementin one or more of those medical conditions. For instance, a subject mayexhibit at least about 5%, at least about 10%, at least about 20%, or atleast about 50% improvement in the clinical symptoms of neuropathy orhyperalgesia compared to the symptoms as assessed prior toadministration of the aromatic-cationic peptide. In some embodiments, asubject may exhibit at least about 5%, at least about 10%, at leastabout 20%, or at least about 50% reduction in the duration of symptomsof neuropathy or hyperalgesia. In some embodiments, a subject mayexhibit at least about 5%, at least about 10%, at least about 20%, or atleast about 50% reduction in the frequency of symptoms of neuropathy orhyperalgesia. In some embodiments, a subject may exhibit at least about5%, at least about 10%, at least about 20%, or at least about 50%reduction in the severity of symptoms of neuropathy or hyperalgesia. Insome embodiments, the subject may show improvement in one or more of theduration, frequency, or severity of symptoms of neuropathy orhyperalgesia.

Combination Therapies

The present disclosure contemplates combination therapies comprising theadministration of an aromatic-cationic peptide such asD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ with one or more additionaltherapeutic regimens. In some embodiments, the additional therapeuticregimens are directed to the treatment or prevention of neuropathy orhyperalgesia or symptoms associated with neuropathy or hyperalgesia. Insome embodiments, the additional therapeutic regimens are directed tothe treatment or prevention of diseases or medical conditions unrelatedto neuropathy or hyperalgesia. In some embodiments, the additionaltherapeutic regimens include regimens directed to the treatment orprevention of neuropathy or hyperalgesia or symptoms associated withneuropathy or hyperalgesia, in addition to diseases, medical conditions,or symptoms unrelated to neuropathy or hyperalgesia or symptomsassociated with neuropathy or hyperalgesia. In some embodiments, theadditional therapeutic regimens comprise administration of one or moredrugs, including, but not limited to, anti-microbials, anti-neoplasticagents, cardiovascular drugs, hypnotics and psychotropics,anti-rheumatics, and anti-convulsants. In embodiments, the additionaltherapeutic regimens comprise non-pharmaceutical therapies, including,but not limited to, dietary and lifestyle management.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vivo methods typically includethe administration of an aromatic-cationic peptide, such as thosedescribed above, to a mammal, such as a human. When used in vivo fortherapy, the aromatic-cationic peptides of the present technology areadministered to the subject in effective amounts (i.e., amounts thathave desired therapeutic effect). They will normally be administeredintravenously, orally, subcutaneously, transdermally, intraperitoneally,intrathecally intramuscularly, intranasally, bucally, sublingually,translingually, or topically. The dose and dosage regimen will dependupon the severity of neuropathy, the characteristics of the particulararomatic-cationic peptide used, e.g., its therapeutic index, thesubject, and the subject's history.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide, such as in a pharmaceutical composition,may be administered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Thepeptide may be administered systemically or locally.

The peptide may be formulated as a pharmaceutically acceptable salt. Theterm “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,acetate or trifluoroacetate, and the like.

The aromatic-cationic peptides described herein can be incorporated intopharmaceutical compositions for administration, singly or incombination, to a subject for the treatment or prevention of a disorderor medical condition described herein. Such compositions typicallyinclude the active agent and a pharmaceutically acceptable carrier. Asused herein the term “pharmaceutically acceptable carrier” includessaline, solvents, dispersion media, coatings, anti-bacterial andanti-fungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;anti-bacterial agents such as benzyl alcohol or methyl parabens;anti-oxidants such as ascorbic acid or sodium bisulfite; chelatingagents such as ethylenediaminetetraacetic acid; buffers such asacetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The aromatic-cationic peptide compositions can include a carrier, whichcan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various anti-bacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thiomersal, and the like. In many cases, it will bedesirable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art. In one embodiment, transdermaladministration may be performed my iontophoresis.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to other cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the present technology, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptides,sufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. In some embodiments, the dosageranges are from about 0.0001 mg per kilogram body weight per day toabout 100 mg per kilogram body weight per day. For example, dosages canbe 1 mg/kg body weight or 10 mg/kg body weight every day, every two daysor every three days or within the range of 1-10 mg/kg every week, everytwo weeks or every three weeks. In one embodiment, a single dosage ofpeptide ranges from 0.1-10,000 micrograms per kg body weight. In oneembodiment, aromatic-cationic peptide concentrations in a carrier rangefrom 0.2 to 2000 micrograms per delivered milliliter. An exemplarytreatment regime entails administration once per day or once a week.Thereafter, the patient can be administered a prophylactic regime.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g., approximately 10⁻⁷molar. This concentration may be delivered by systemic doses of 0.01 to100 mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, such as by single daily or weekly administration, butalso including continuous administration (e.g., parenteral infusion ortransdermal application).

In some embodiments, the dosage of the aromatic-cationic peptide isprovided at a “low,” “mid,” or “high” dose level. In one embodiment, thelow dose is provided from about 0.001 to about 0.5 mg/kg/h, suitablyfrom about 0.01 to about 0.1 mg/kg/h. In one embodiment, the mid-dose isprovided from about 0.1 to about 1.0 mg/kg/h, suitably from about 0.1 toabout 0.5 mg/kg/h. In one embodiment, the high dose is provided fromabout 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2mg/kg/h.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject,including, but not limited to, the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance with the present technology can be anymammal, including, for example, farm animals, such as sheep, pigs, cows,and horses; pet animals, such as dogs and cats; laboratory animals, suchas rats, mice and rabbits. In a some embodiments, the mammal is a human.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1: Use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in thePrevention and Treatment of Drug-Induced Hyperalgesia in Rats

This example illustrates the methods and compositions of the presenttechnology in the prevention and treatment of hyperalgesia. The exampledemonstrates the use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in theprevention and treatment of vincristine-induced hyperalgesia in rats.

Female Sprague Dawley rats were divided randomly into 5 groups (n=10),according to Table 7. Groups 2-4 were administered Vincristine 0.10mg/kg injected intravenously (i.v.) via tail vein followed by a bolusinjection of 0.1 ml PBS vehicle, once daily for 14 days. Group 1 wasadministered 1 ml/kg PBS vehicle alone according to the same schedule.D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ 10 mg/kg was administeredsubcutaneously (s.c.) 15 minutes prior to vincristine administration forthe full 14-day period (Group 3), or for a period of two weeks beginningat day 15 (Group 4). Group 5 was administered Vincristine 0.10 mg/kginjected i.v. via tail vein, followed by a bolus injection of 0.1 ml PBSvehicle once daily for 28 days, andD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ 10 mg/kg administered s.c. for aperiod of 14 days beginning at day 15.

TABLE 7 Use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in the Preventionand Treatment of Drug-Induced Hyperalgesia in Rats Group ConditionTreatment* 1 Untreated Control PBS vehicle 2 PBS Control Vincristine +PBS vehicle 3 D-Arg-2′6′- Vincristine + D-Arg-2′6′-dimethyltyrosine-dimethyltyrosine- Lys-Phe-NH₂ Lys-Phe-NH₂ 4 D-Arg-2′6′- (i) Vincristine(14 days) dimethyltyrosine- (ii) D-Arg-2′6′-dimethyltyrosine-Lys-Phe-Lys-Phe-NH₂ NH₂, beginning at day 15 5 D-Arg-2′6′- (i) Vincristine (28days) dimethyltyrosine- (ii) D-Arg-2′6′-dimethyltyrosine-Lys-Phe-Lys-Phe-NH₂ NH₂, beginning at day 15 *Vincristine 0.10 mg/kg injecteddaily i.v.; D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ 10 mg/kg injecteddaily s.c.; PBS vehicle at pH 7.0; Von Frey test conducted every twodays

Mechanical nociceptive threshold of paw-withdrawal was measured everytwo days using an electronic Von Frey test. Results are shown in FIG. 1and FIG. 2 as means±SEM. One-way ANOVA with post-hoc test or t-test wereused to assess the statistical significance, with p<0.05 is consideredstatistically significant. In FIG. 1, data for subjects receivingvincristine in the absence of simultaneousD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ (Groups 2, 4, 5) is showncollectively as “Group A.”

Results—

As shown in FIG. 1, subjects administered vincristine for a period of 14days (Group A) showed a decrease in pain threshold compared to avehicle-only control (Group 1). By contrast, the pain threshold ofsubjects administered D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ togetherwith vincristine (Group 3) was not significantly different from thevehicle-only control. This shows that co-administration ofD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ together with vincristineprevents vincristine-induced hyperalgesia in mammalian subjects.

As shown in FIG. 2, subjects administered vincristine for a period of 14days (Groups 2, 4) showed a decrease in pain threshold response comparedto a vehicle-only control (Group 1). Following the withdrawal ofvincristine, subjects not administeredD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ continued to show a decline inpain threshold, reaching a low point at day 21, and showed a moderateimprovement beginning at day 22 (Group 2). By contrast, subjectsadministered D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ following thewithdrawal of vincristine showed improved pain threshold as soon as day15, with continued improvement through day 25 (Group 4). This shows thatadministration of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ subsequent tovincristine therapy improves the rate of recovery of a mammalian subjectfrom vincristine-induced hyperalgesia.

As further shown in FIG. 2, subjects administered vincristine in theabsence of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ for a period of 14days, and then administered vincristine together withD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ for a period of 14 days, showedan arrest of the downward trend in pain threshold evident in the initial14 day period, followed by an improvement in pain threshold beginning atday 16 (Group 5). This shows that administration ofD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ subsequent to the development ofvincristine-induced hyperalgesia is effective in halting furtherprogression of the condition and promotes improvement of the condition,even where a subject continues to receive vincristine.

This example shows that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂, are usefulin the prevention and treatment of vincristine-induced hyperalgesia. Theresults further show that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ are usefulin the prevention and treatment of drug-induced hyperalgesia generally.

Example 2—Use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in theTreatment of Drug-Induced Hyperalgesia in Humans

This example will demonstrate use of the methods and compositions of thepresent technology in the treatment of hyperalgesia in human subjects.The example will demonstrate the use ofD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in the treatment ofvincristine-induced hyperalgesia in humans.

Patients will be recruited to the study as they present in clinic withchronic (>6 months' duration), spontaneous, ongoing, vincristine-relatedpain. Those enrolled will rate their daily maximum level of pain at 4 orgreater on a visual analog scale (VAS). The patients will be screenedfor their willingness to enroll in the study, and informed consent willbe obtained. Healthy subjects will also be recruited for collection ofcomparison data. No subjects in either the patient or comparison groupwill have known risk factors for any other cause of peripheralneuropathy, including diabetes, AIDS, chronic alcoholism, or previousradiation exposure.

After a focused interview about the history of the patient's cancer andtreatment, the patient will be asked to describe sensory symptoms bychoosing from a list of ideal type word descriptors. Ongoing and dailymaximum pain intensity will be rated on a VAS with prompts of “no pain”at the bottom and “most imaginable” at the top. The areas of pain andsensory disturbances will be drawn by each patient on a standardizedbody map. Similar to previous observations in patients treated withpaclitaxel, subjects with vincristine-induced peripheral neuropathy arepredicted to identify the following three zones of sensation:

-   -   a) The painful area: The zone of ongoing pain located on the        tips of the fingers and/or toes. The tip of the index finger is        expected to be involved in all patients and will be used as the        test site in this zone.    -   b) The border area: Adjacent and proximal to, but distinct from        the painful area, represented by nonpainful sensory disturbances        and located in the palms and/or soles of the feet. The thenar        eminence is expected to be involved in all patients and will be        used as the test site in this zone.    -   c) The nonpainful area: Adjacent and proximal to, but distinct        from the border area, reported by the patient to feel “normal.”        This site is expected to be always proximal to the wrists and/or        ankles. Sensory testing will be conducted on the volar surface        of the arm.

The tip of the index finger, thenar eminence, and volar forearm, will betested in normal subjects for comparison. Patients will be specificallyqueried about the stimuli that provoked pain or caused an exacerbationof ongoing pain in these regions, including the effects that clothing,bed linens, bathing, and normal activities of daily living cause. Eachzone will be examined for any physical changes, such as scaling, fingerclubbing, and erythema, which will be documented. The areas of sensorydisturbance will be physically probed by light touch with a camel hairbrush and by manual massage to screen for the presence of allodynia orhyperalgesia.

Touch and Sharpness Detection Thresholds—

Touch detection thresholds will be determined with von Freymonofilaments using the up/down method as previously reported. Startingwith a bending force of 0.02 g, each monofilament will be applied to aspot on the skin less than 2 mm in diameter for approximately onesecond. The force of the filament detected four consecutive times willbe assigned as the touch detection threshold. Sharpness detection willbe determined using weighted 30-gauge metal cylinders. Briefly, the tipof 30-gauge needles (200 mm diameter) will be filed to produce flat,cylindrical ends and the luers will be fitted to calibrated brassweights with the desired force (100, 200, and 400 mN) level for eachstimulus. Each loaded needle will be placed inside a separate 10 ccsyringe where it will be able to move freely. Each stimulus will beapplied for one second perpendicular to the skin 10 times within eacharea of interest in a pseudorandom order. The subjects will indicatewhether the stimulus is perceived as touch, pressure, sharp, or other.The percentages of each reply will be calculated and then combined intogroup grand means for comparison. The 50% sharpness detection thresholdwill be calculated as the weighted needle that caused five or more sharpresponses after 10 consecutive stimuli.

Grooved Pegboard Test—

Manual dexterity will be assessed with the grooved pegboard test.Subjects will be instructed to fill a five-by-five slotted pegboard inan ordered fashion and the times for both dominant and non-dominanthands will be recorded.

Thermal Detection Thresholds—

The threshold for heat pain will be determined using the Marstocktechnique. A radiometer will be used at the outset of testing toascertain the baseline skin temperature at all testing sites. All testsand measurements will be conducted at room temperature 22° C. Thermalramps will be applied using a 3.6×3.6 cm Peltier thermode from abaseline temperature of 32° C. Skin heating will be at a ramp of 0.30°C./s, and skin cooling will be at a ramp of −0.5° C./s. Subjects will beinstructed to signal when the stimulus is perceived as first becomingwarmer and then painfully hot, or as first becoming cooler and thenpainfully cold. If a subject fails to reach a given threshold before thecutoff temperature of 51.5° C. for the ascending ramp or 3° C. held for10 seconds in the cooling test, the cutoff values will be assigned forany that are not reached. The final threshold value for each skinsensation in each patient will be determined by averaging the results ofthree heating and cooling trials.

Statistical Analysis—

The thresholds for touch detection will be compared using nonparametricmethods (Wilcoxon's test). The sharpness detection, thermal thresholds,and times in the grooved pegboard tests will be compared using analysisof variance and post hoc comparison of the means with Duncan's multiplerange tests. Comparisons of mechanical and thermal thresholds will beperformed between healthy subjects and patients for the different areasof the tested skin. Further analyses will be performed between glabrousand volar skin within the patient group. For every comparison performedin the present study, p<0.05 will be considered significant.

Following initial assessment of the above criteria, subjects will bedivided into four groups:

-   -   a) Healthy controls    -   b) No treatment    -   c) Vehicle-only placebo, administered s.c., once daily for 14        days    -   d) D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ at 1-100 mg/kg (e.g.,        1 mg/kg, 5 mg, kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50        mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg or 100 mg/kg)        administered s.c., once daily for 14 days

Following the 14 day treatment period, subjects will be re-assessedaccording to the above criteria, with statistical analysis as describedabove.

Results—

It is expected that neuropathy subjects administeredD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ for a period of 14 days willreport a reduction in hyperalgesia symptoms compared to subjectsadministered no treatment or a vehicle-only placebo. The reduction inhyperalgesia will be manifest in improved scoring for touch andsharpness detection thresholds, grooved pegboard tests, and thermaldetection tests compared to control subjects.

These results will show that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ are usefulin the treatment of vincristine-induced hyperalgesia, and drug-inducedhyperalgesia generally. The results will show that the methods andcompositions described herein are useful in the treatment ofdrug-induced peripheral neuropathy or hyperalgesia.

Example 3—Use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in theTreatment of Hyperalgesia in Humans

This example will demonstrate use of the methods and compositions of thepresent technology in the treatment of hyperalgesia. The example willdemonstrate the use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in thetreatment of hyperalgesia associated with peripheral neuropathy ofvarious etiologies in humans.

Patients will be recruited to the study as they present in clinic withchronic (>6 months' duration), spontaneous, ongoing, neuropathy-relatedpain. Independent studies will address neuropathies resulting from,caused by, or otherwise associated with genetic disorders,metabolic/endocrine complications, inflammatory diseases, vitamindeficiencies, malignant diseases, and toxicity, such as alcohol, organicmetal, heavy metal, radiation, and drug toxicity. Subjects will beselected such that they have a single type of neuropathy and no knownrisk factors for neuropathy types outside the scope of the study inwhich the subject is enrolled. Those enrolled will rate their dailymaximum level of pain at 4 or greater on a visual analog scale (VAS).Subjects will be screened for their willingness to enroll in the study,and informed consent will be obtained. Healthy subjects will also berecruited for collection of comparison data.

After a focused interview about the medical history, the patient will beasked to describe sensory symptoms by choosing from a list of ideal typeword descriptors. Ongoing and daily maximum pain intensity will be ratedon a VAS with prompts of “no pain” at the bottom and “most imaginable”at the top. The areas of pain and sensory disturbances will be drawn byeach patient on a standardized body map. Neuropathy subjects arepredicted to identify the following three zones of sensation:

-   -   a) The painful area: The zone of ongoing pain located on the        tips of the fingers and/or toes. The tip of the index finger is        expected to be involved in all patients and will be used as the        test site in this zone.    -   b) The border area: Adjacent and proximal to, but distinct from        the painful area, represented by nonpainful sensory disturbances        and located in the palms and/or soles of the feet. The thenar        eminence is expected to be involved in all patients and will be        used as the test site in this zone.    -   c) The nonpainful area: Adjacent and proximal to, but distinct        from the border area, reported by the patient to feel “normal.”        This site is expected to be always proximal to the wrists and/or        ankles. Sensory testing will be conducted on the volar surface        of the arm.

The tip of the index finger, thenar eminence, and volar forearm, will betested in normal subjects for comparison. Patients will be specificallyqueried about the stimuli that provoked pain or caused an exacerbationof ongoing pain in these regions, including the effects that clothing,bed linens, bathing, and normal activities of daily living cause. Eachzone will be examined for any physical changes, such as scaling, fingerclubbing, and erythema, which will be documented. The areas of sensorydisturbance will be physically probed by light touch with a camel hairbrush and by manual massage to screen for the presence of allodynia orhyperalgesia.

Touch and Sharpness Detection Thresholds—

Touch detection thresholds will be determined with von Freymonofilaments using the up/down method as previously reported. Startingwith a bending force of 0.02 g, each monofilament will be applied to aspot on the skin less than 2 mm in diameter for approximately onesecond. The force of the filament detected four consecutive times willbe assigned as the touch detection threshold. Sharpness detection willbe determined using weighted 30-gauge metal cylinders. Briefly, the tipof 30-gauge needles (200 mm diameter) will be filed to produce flat,cylindrical ends and the luers will be fitted to calibrated brassweights with the desired force (100, 200, and 400 mN) level for eachstimulus. Each loaded needle will be placed inside a separate 10 ccsyringe where it will be able to move freely. Each stimulus will beapplied for one second perpendicular to the skin 10 times within eacharea of interest in a pseudorandom order. The subjects will indicatewhether the stimulus is perceived as touch, pressure, sharp, or other.The percentages of each reply will be calculated and then combined intogroup grand means for comparison. The 50% sharpness detection thresholdwill be calculated as the weighted needle that caused five or more sharpresponses after 10 consecutive stimuli.

Grooved Pegboard Test—

Manual dexterity will be assessed with the grooved pegboard test.Subjects will be instructed to fill a five-by-five slotted pegboard inan ordered fashion and the times for both dominant and non-dominanthands will be recorded

Thermal Detection Thresholds—

The threshold for heat pain will be determined using the Marstocktechnique. A radiometer will be used at the outset of testing toascertain the baseline skin temperature at all testing sites. All testsand measurements will be conducted at room temperature 22° C. Thermalramps will be applied using a 3.6×3.6 cm Peltier thermode from abaseline temperature of 32° C. Skin heating will be at a ramp of 0.30°C./s, and skin cooling will be at a ramp of −0.5° C./s. Subjects will beinstructed to signal when the stimulus is perceived as first becomingwarmer and then painfully hot, or as first becoming cooler and thenpainfully cold. If a subject fails to reach a given threshold before thecutoff temperature of 51.5° C. for the ascending ramp or 3° C. held for10 seconds in the cooling test, the cutoff values will be assigned forany that are not reached. The final threshold value for each skinsensation in each patient will be determined by averaging the results ofthree heating and cooling trials.

Statistical Analysis—

The thresholds for touch detection will be compared using nonparametricmethods (Wilcoxon's test). The sharpness detection, thermal thresholds,and times in the grooved pegboard tests will be compared using analysisof variance and post hoc comparison of the means with Duncan's multiplerange tests. Comparisons of mechanical and thermal thresholds will beperformed between healthy subjects and patients for the different areasof the tested skin. Further analyses will be performed between glabrousand volar skin within the patient group. For every comparison performedin the present study, p<0.05 will be considered significant.

Following initial assessment of the above criteria, subjects will bedivided into four groups:

-   -   a) Healthy controls    -   b) No treatment    -   c) Vehicle-only placebo, administered s.c., once daily for 14        days    -   d) D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ at 1-100 mg/kg (e.g.,        1 mg/kg, 5 mg/kg, kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50        mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg or 100 mg/kg)        administered s.c., once daily for 14 days

Following the 14 day treatment period, subjects will be re-assessedaccording to the above criteria, with statistical analysis as describedabove.

Results—

It is expected that neuropathy subjects administeredD-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ for a period of 14 days willreport a reduction in hyperalgesia compared to subjects administered avehicle-only placebo. The reduction in hyperalgesia will be manifest inimproved scoring for touch and sharpness detection thresholds, groovedpegboard tests, and thermal detection tests compared to controlsubjects.

These results will show that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂, are usefulin the treatment of neuropathy-related hyperalgesia generally.

Example 4—Use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in thePrevention of Hyperalgesia in Humans

This example will demonstrate use of the methods and compositions of thepresent technology in the prevention of hyperalgesia. The example willdemonstrate the use of D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ in theprevention of hyperalgesia associated with peripheral neuropathy ofvarious etiologies in humans.

Subjects at risk for developing hyperalgesia will be recruited as theypresent in clinic for the treatment of conditions associated with thedevelopment of peripheral neuropathy or hyperalgesia. Independentstudies will address neuropathy and hyperalgesia resulting from, causedby, or otherwise associated with genetic disorders, metabolic/endocrinecomplications, inflammatory diseases, vitamin deficiencies, malignantdiseases, and toxicity, such as alcohol, organic metal, heavy metal,radiation, and drug toxicity. Subjects will be selected such that theyare at risk for developing a single type of neuropathy or hyperalgesia,having no risk factors outside the scope of the study in which thesubject is enrolled, and as yet not having symptoms associated withneuropathy or hyperalgesia. Subjects will be screened for theirwillingness to enroll in the study, and informed consent will beobtained. Healthy subjects will also be recruited for collection ofcomparison data.

After a focused interview about the medical history, baselinemeasurements of touch and sharpness detection thresholds, groovedpegboard tests, and thermal detection thresholds will be determinedaccording to the methods described above, with statistical analysis asdescribed above.

Following initial assessment of the above criteria, subjects will bedivided into four groups:

-   -   a) Healthy controls    -   b) No treatment    -   c) Vehicle-only placebo, administered s.c., once daily    -   d) D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ at 1-100 mg/kg (e.g.,        1 mg/kg, 5 mg/kg, kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50        mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg or 100 mg/kg)        administered s.c., once daily

Subjects will be evaluated weekly during the trial for sharpnessdetection thresholds, grooved pegboard tests, and thermal detectionthresholds. The trial will continue for a period of 28 days, or untilthe no-treatment and placebo control groups display hyperalgesiaaccording to the above criteria, at which point subjects will undergo afinal assessment.

Results—

It is expected that subjects at risk of developing neuropathy orhyperalgesia administered D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂ willshow attenuated development of neuropathy or hyperalgesia compared tountreated and placebo controls.

These results will show that aromatic-cationic peptides of the presenttechnology, such as D-Arg-2′6′-dimethyltyrosine-Lys-Phe-NH₂, are usefulin the prevention of neuropathy and hyperalgesia generally. The resultswill show that the methods and compositions described herein are usefulin the prevention of neuropathy or hyperalgesia generally.

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of the present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andcompositions within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presenttechnology is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this technology is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All references cited herein are incorporated herein by reference intheir entireties and for all purposes to the same extent as if eachindividual publication, patent, or patent application was specificallyand individually incorporated by reference in its entirety for allpurposes.

Other embodiments are set forth within the following claims.

1.-30. (canceled)
 31. A method for treating peripheral neuropathy orhyperalgesia in a subject in need thereof, comprising administering tothe subject an effective amount of a peptide having the formulaPhe-D-Arg-Phe-Lys-NH₂.
 32. The method of claim 31, wherein theperipheral neuropathy or hyperalgesia is drug-induced.
 33. The method ofclaim 32, wherein the drug is a chemotherapeutic agent.
 34. The methodof claim 33, wherein the chemotherapeutic agent is procarbazine,nitrofurazone, podophyllum, mustine, ethoglucid, cisplatin, suramin,paclitaxel, chlorambucil, altretamine, carboplatin, cytarabine,docetaxel, dacarbazine, etoposide, ifosfamide with mesna, fludarabine,tamoxifen, teniposide, thioguanine, or vincristine.
 35. The method ofclaim 33, wherein the chemotherapeutic agent is vincristine.
 36. Themethod of claim 32, wherein the peptide is administered simultaneouswith the drug.
 37. The method of claim 32, wherein the peptide isadministered subsequent to the drug.
 38. The method of claim 31, whereinthe peripheral neuropathy causes hyperalgesia.
 39. The method of claim31, wherein the subject is a human.
 40. The method of claim 31, whereinthe peptide is administered intravenously, orally, subcutaneously,transdermally, intraperitoneally, intrathecally intramuscularly,intranasally, bucally, sublingually, translingually, or topically.
 41. Amethod for preventing hyperalgesia in a subject in need thereof,comprising administering to the subject an effective amount of a peptidehaving the formula Phe-D-Arg-Phe-Lys-NH₂.
 42. The method of claim 41,wherein the hyperalgesia is drug-induced.
 43. The method of claim 42,wherein the drug is a chemotherapeutic agent.
 44. The method of claim43, wherein the chemotherapeutic agent is procarbazine, nitrofurazone,podophyllum, mustine, ethoglucid, cisplatin, suramin, paclitaxel,chlorambucil, altretamine, carboplatin, cytarabine, docetaxel,dacarbazine, etoposide, ifosfamide with mesna, fludarabine, tamoxifen,teniposide, thioguanine, or vincristine.
 45. The method of claim 43,wherein the chemotherapeutic agent is vincristine.
 46. The method ofclaim 42, wherein the peptide is administered simultaneous with thedrug.
 47. The method of claim 42, wherein the peptide is administeredsubsequent to the drug.
 48. The method of claim 41, wherein the peptideis administered prior the onset of hyperalgesia.
 49. The method of claim41, wherein the subject is a human.
 50. The method of claim 41, whereinthe peptide is administered intravenously, orally, subcutaneously,transdermally, intraperitoneally, intrathecally intramuscularly,intranasally, bucally, sublingually, translingually, or topically.